The innate host response to bacterial pathogens is characterized by an immediate release of biologically active compounds, including monokines and cytokines. These proinflammatory molecules, which are intended to enable the host to eliminate the pathogen, may also adversely affect the host. In acute situations, the pathogen is often eliminated, with resolution of inflammation and minimal tissue damage. However, failure to control the pathogen often leads to a state of metabolic anarchy in which the inflammatory response is not controlled and significant tissue damage results. Endotoxins, produced from the outer membrane of Gram-negative bacteria, and exotoxins, released from the cell wall of Gram-positive bacteria, are known to be potent inducers of the inflammatory response. Lipopolysaccharide (LPS), extracted from the outer membrane of Gram-negative bacteria, has been identified as a principal endotoxic component.
Although the inflammatory response is mediated by a variety of secreted factors, the cytotoxic effects of LPS have been ascribed to TNF-xcex1 activity (Beutler et al., Science 229: 869-871 (1985); Tracey et al., Science 234: 470-474 (1986); Miethke et al., J. Exp. Med. 175: 91-98 (1992)). TNF-xcex1 is a pleiotropic cytokine which serves to either benefit the host or in some situations exert detrimental effects on the host (Beutler and Cerami, Nature 320: 584-588 (1986); Beutler et al., Science 232: 977-980 (1986); Beutler and Cerami, N. Engl. J. Med. 316: 379-385 (1987)). TNF-xcex1 benefits the host by helping to prevent cancer, protecting against infection, promoting tissue remodeling, and activating inflammatory responses. Conversely, in host responses which have gone awry, TNF-xcex1 mediates septic shock in chronic infections, is responsible for cachexia in cancer patients, causes inflammation in rheumatoid arthritis patients, and activates the human immunodeficiency virus. The pleiotropic effects of TNF-xcex1 are dose-dependent. Hence, the perceived need to control TNF-xcex1 production has raised interest into the understanding of the mechanisms that modulate TNF-xcex1 gene expression.
It is well known that gene transcription is controlled by DNA-binding proteins. Recently, several groups have examined the transcriptional regulation of TNF-xcex1 by various inducers, such as virus, LPS, and PMA. The human TNF-xcex1 promoter contains motifs that resemble nuclear factor kappa B (NF-xcexaB) binding sites; however, controversy exists as to the involvement of NF-xcexaB in TNF-xcex1 gene regulation. The nature of the nuclear factor(s) involved in the regulation of LPS-induced TNF-xcex1 gene expression in humans remains unknown.
In one aspect, the present invention relates to an isolated polypeptide which binds to the DNA binding domain located from xe2x88x92550 to xe2x88x92487 in the promoter of the human TNF-xcex1 gene. This isolated polypeptide is referred to herein as the LITAF protein. In one embodiment, the isolated polypeptide is mammalian in origin. In a preferred embodiment, it is human or murine.
In another aspect the present invention relates to a nucleic acid sequences which encodes the LITAF protein. Nucleic acid sequences which are characterized by the ability to hybridize to the complement of the nucleic acid sequence of the present invention under stringent hybridization conditions are also encompassed by the present invention. Also encompassed is an expression vector comprising a nucleic acid sequence which encodes the LITAF protein. Also encompassed is a cell containing said expression vector, and a mammalian gene which encodes the LITAF protein. Preferably, the gene is human and is located on Chromosome 16 p12-16p13.3.
In another aspect, the present invention relates to an antibody characterized by the ability to specifically bind to the LITAF protein. The antibody may be monoclonal or polyclonal.
Another aspect of the present invention relates to a method for inhibiting LITAF dependent induction of TNF-xcex1 gene expression in a cell, comprising the steps a) providing an inhibitor of expression of the LITAF gene; and b) delivering the inhibitor into the cell. In one embodiment, the inhibitor is an antisense construct which encodes an antisense RNA molecule which is complementary to a portion of the LITAF mRNA which is greater than 200 nucleotides in length. Preferably, the antisense RNA molecule is complementary to the start site of translation, upstream adjacent 5xe2x80x2 untranslated sequence, and downstream adjacent coding sequence of the LITAF mRNA. Optimal lengths and specific nucleotides for complementary are discussed.
Another aspect of the present invention relates to a method for inhibiting LITAF dependent induction of TNF-xcex1 gene expression in a cell comprising the steps a) providing an inhibitor of LITAF protein function; and b) contacting the inhibitor to the LITAF in the cell. In one embodiment, the inhibitor is an antibody which binds the LITAF protein. In another embodiment the inhibitor is a small molecule which inhibits the function of the LITAF protein. One example of such an inhibitor is a recombinant mutant LITAF protein.
Therapeutic methods for treating a patient with a disease associated with chronic inflammation, by administration of the LITAF inhibitor to the patient are also provided. Such diseases include rheumatoid arthritis, gum disease Crohn""s disease, and graft-versus-host disease. Therapeutic methods for treating a patient with a disease in which TNF-xcex1 plays a role in pathology are also provided. Examples of such diseases are diabetes mellitus, cancer, cachexia, breast cancer, HIV, sepsis, malaria, trypanomiasis and asthma. Other methods provided include a method for identifying gene which are regulated by the LITAF protein, a method for identifying a molecule which inhibits LITAF binding to the TNF-xcex1 promoter, and a method for identifying molecules which bind LITAF from a protein array.